The major challenge to comprehending metalloprotein function is increasingly not data availability but rather the ability to interpret results and effectively apply these advances to solve critical biological problems. This Program seeks to apply the fully synergistic power of a Program Project to bridge this growing gap between detailed description of structures and in depth comprehension of activity for metalloproteins. Metalloprotein activity reflects the complexity results from the structural chemistry of the metal ion and three surrounding protein layers: the ligands (first shell), the ligand-contacting residues and solvent neighborhood (second shell), and the protein environment (third shell). To interpret, this complex relationships, this Program integrates experimental characterization from structure determination, spectroscopy, protein engineering; theoretical and experimental characterizations from structure determination, spectroscopy, protein engineering, theoretical and computational approaches, and computer graphics with experimental testing of functional hypotheses by metalloprotein design. Metalloprotein design, the predetermined placement and alteration of metal centers in proteins, will test and extend comprehension of the structural chemistry underlying metal site affinity, specificity and function. A major focus is to establish a direct experimental correlation between structural design parameters and metalloprotein properties by using an accurate database of protein metal sites, explicit evaluation of possible states, projects that separate general and framework-specific effects, and efficient computational searchers in the vast combinatorial landscape of the metal site environment. A major Program goal is to combined the new green fluorescent protein (GFP) technology, the DEZYMER software for algorithmic design, and the Quantitative Metalloprotein Database to join the advantages of structure-based design with the power for comprehensive genetic screening. Resulting GFP metallomutants promise to provide biologically useful metal ion sensors within cells. Together these projects will investigate the major types of metal sites in proteins (Fe, di-Fe, Fe in heme and FeS clusters, Cu, Zn, Mn, and Ca sites), and their results will be synergistic for understanding common and variable features of metalloprotein structure and function. Overall, we seek to develop, test, and apply rules governing metal site coordination, affinity and activity in proteins, by using both theory and experiment. The ability to understand and ultimately control the binding and activity of protein metal sites, which are required by about one-third of all proteins, is of great medical and biological importance.